Abstract. Neural circuit function requires synaptic inhibition mediated by ?-aminobutyric acid (GABA). Glutamine is the major metabolic precursor for neuronal GABA synthesis and is supplied to neurons via the activity of the astrocyte-specific enzyme glutamine synthetase (GS). Inhibition or brain-specific genetic ablation of GS leads to impaired GABAergic inhibition, intractable epilepsy, and death. Consistent with this, deficits in GS expression lead to epilepsy and are implicated in neurodegeneration. However, to date, there have been no systematic studies to evaluate how GS activity is regulated to meet the demand of neurons for glutamine. Here we will address the role that cAMP-dependent phosphorylation plays in regulating GS activity, and if this process contributes to the deficits in GABAergic inhibition that result in epilepsy. To do so, we will identify sites of phosphorylation within GS, under control conditions and during Status Epilepticus (SE), the most severe form of epilepsy and a medical emergency. The effects that phosphorylation have on GS activity will then be determined using high-resolution enzyme kinetics. The cellular mechanisms that regulate GS phosphorylation will be explored using Designer Receptors Exclusively Activated by Designer Drugs (DREADD) to selectively modulate PKA signaling in astrocytes. Finally, we will assess the significance of GS phosphorylation for the efficacy of GABAergic inhibition using viral expression to replace endogenous GS molecules with mutants in which phosphorylation of critical regulatory residues has been prevented. Preliminary studies have allowed us to formulate a central hypothesis that will be tested here: GS activity is negatively regulated by PKA- dependent phosphorylation, and this process contributes to the deficits in GABAergic inhibition that are fundamental to the pathophysiology of epilepsy. Our proposal will center on the following specific aims: Aim 1. To test the hypothesis that PKA-mediated phosphorylation of GS leads to decreased enzyme activity. Aim 2. To test the hypothesis that phospho-dependent inactivation of GS is enhanced by seizure activity. Aim 3. To test the hypothesis that GS phosphorylation contributes to the deficits in GABAergic inhibition during SE. Collectively, these studies will provide the first evidence that GS is subject to phospho-dependent modulation and that this process contributes to the deficits in GABAergic inhibition seen in SE. Such insights may lead to improved therapies to reduce the impact of epilepsy. !